This study screened four Rhizopus species as biosurfactant producers using different markers. First of all, Rhizopus spp. UCP 1607 was identified as Rhizopus arrhizus by morphological and molecular methods. The production of biosurfactant/bioemulsifier was investigated by submerged fermentation using soybean post-frying oil (5% v/v) and sodium glutamate (1% w/v) medium. The primary markers' hemolysis and parafilm M tests showed that R. arrhizus UCP1607 strain exhibited higher hemolytic activity (49 mm of clear zone) on sheep blood agar and a larger drop diameter (12 mm) on parafilm hydrophobic surface. The experimental results showed the most promising biosurfactant production by R. arrhizus UCP 1607 strain led to a reduction of surface tension (31.8 mN/m) and the diameter of the oil-spreading covered an area of 66.4 cm 2 . The strains Rhizopus microsporus var. chinensis UCP1296, R. microsporus var. microsporus UCP1304, and R. arrhizus UCP1607 were capable of forming stable emulsions corresponding to 91.7, 94.8, and 82.6%, respectively in crude oil.